Figure 4.
KYNU and SF3B3 are the main self-ligands of 2F5 and 4E10. Inhibition of 2F5 binding to immobilized recombinant KYNU (A) or CMTM3 (B) assessed in ELISA as described in Materials and methods. The y-axis indicates OD percentage of maximal binding, which is determined as the mean reading without inhibitors (100% binding is marked by gray dashes for comparison). The x-axis designates the molar ratio of inhibitor to plate immobilized ligand. (C) 2F5 and 151K immunoprecipitation eluates were stained with KYNU Ab in Western blot. Recombinant KYNU (rKYNU) was used as a positive control; molecular masses (kD) of protein markers are shown on the left of gel. (D and E) HEp-2 slides were indirectly labeled with anti-KYNU (FITC, green) and 2F5 (PE, red; D) or 4E10 (FITC, green) and anti-SF3B3 (PE, red; E). Negative controls were included to ensure specific staining and minimal cross-reactivity of all primary and secondary Abs. Bars, 15 µm. All data are representative of three or more independent experiments.